title: Exon skipping as a therapeutic strategy applied to an RYR1 mutation with pseudo-exon inclusion causing a severe core myopathy.
abstract: Central core disease is a myopathy often arising from mutations in the type 1 ryanodine receptor (RYR1) gene, encoding the sarcoplasmic reticulum calcium release channel RyR1. No treatment is currently available for this disease. We studied the pathological situation of a severely affected child with two recessive mutations, which resulted in a massive reduction in the amount of RyR1. The paternal mutation induced the inclusion of a new in-frame pseudo-exon in RyR1 mRNA that resulted in the insertion of additional amino acids leading to the instability of the protein. We hypothesized that skipping this additional exon would be sufficient to restore RyR1 expression and to normalize calcium releases. We therefore developed U7-AON lentiviral vectors to force exon skipping on affected primary muscle cells. The efficiency of the exon skipping was evaluated at the mRNA level, at the protein level, and at the functional level using calcium imaging. In these affected cells, we observed a decreased inclusion of the pseudo-exon, an increased RyR1 protein expression, and a restoration of calcium releases of normal amplitude either upon direct RyR1 stimulation or in response to membrane depolarization. This study is the first demonstration of the potential of exon-skipping strategy for the therapy of central core disease, from the molecular to the functional level.
authors.affiliation: INSERM U836, Grenoble Institut des Neurosciences, Equipe Muscle et Pathologies, 38000 Grenoble, France.
authors.forename: John
authors.initials: J
authors.lastname: Rendu
authors.forename: Julie
authors.initials: J
authors.lastname: Brocard
authors.forename: Eric
authors.initials: E
authors.lastname: Denarier
authors.forename: Nicole
authors.initials: N
authors.lastname: Monnier
authors.forename: France
authors.initials: F
authors.lastname: Piétri-Rouxel
authors.forename: Cyriaque
authors.initials: C
authors.lastname: Beley
authors.forename: Nathalie
authors.initials: N
authors.lastname: Roux-Buisson
authors.forename: Brigitte
authors.initials: B
authors.lastname: Gilbert-Dussardier
authors.forename: Marie José
authors.initials: MJ
authors.lastname: Perez
authors.forename: Norma
authors.initials: N
authors.lastname: Romero
authors.forename: Luis
authors.initials: L
authors.lastname: Garcia
authors.forename: Joël
authors.initials: J
authors.lastname: Lunardi
authors.forename: Julien
authors.initials: J
authors.lastname: Fauré
authors.forename: Anne
authors.initials: A
authors.lastname: Fourest-Lieuvin
authors.forename: Isabelle
authors.initials: I
authors.lastname: Marty
mesh_terms: D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism; D017931:DNA Primers / Q000235:genetics; D005091:Exons / Q000235:genetics*; D005786:Gene Expression Regulation / Q000235:genetics*; D015316:Genetic Therapy / Q000379:methods*; D005822:Genetic Vectors / Q000235:genetics; D057809:HEK293 Cells; D006801:Humans; D016086:Lentivirus; D008856:Microscopy, Fluorescence; D009154:Mutation / Q000235:genetics; D020512:Myopathy, Central Core / Q000628:therapy*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D019837:Ryanodine Receptor Calcium Release Channel / Q000235:genetics* / Q000378:metabolism
chemical_list: D017931:DNA Primers; D019837:Ryanodine Receptor Calcium Release Channel; D002118:Calcium